CHARACTERIZATION AND GENETIC COMPLEMENTATION OF A BRUCELLA-ABORTUS HIGH-TEMPERATURE-REQUIREMENT-A (HTRA) DELETION MUTANT

In order to evaluate the biological function of the Brucella abortus h igh-temperature-requirement A (HtrA) stress response protein homolog, the majority of the htrA gene was deleted from the chromosome of B. ab ortus 2308 via gene replacement. In contrast to the parental strain, t he resulting htrA deletion mutant, designated PHE1, failed to grow on solid medium at 40 degrees C and demonstrated increased sensitivity to killing by H2O2 and O-2(-) in disk sensitivity assays. BALB/c mice we re infected with strains 2308 and PHE1 to assess the effect of the htr A mutation on virulence, and significantly fewer brucellae were recove red from the spleens of mice infected with PNE1 than from those of mit e infected with 2308 at 1 week postinfection, Genetic complementation studies were performed to confirm the relationship between the htrA mu tation and the phenotype observed for PHE1. Plasmid pRIE1 was construc ted by inserting a 1.9-kb EcoRI fragment encoding the B. abortus htrA gene into the broad-host-range plasmid pBBR1MCS. Introduction of pRIE1 into PHE1 relieved the temperature- and H2O2 sensitive phenotypes of this mutant in vitro, and PHE1(pRIE1) colonized. the spleens of BALB/c mice at levels equivalent to those of the parental 2308 strain at 1 w eek postinfection, These results support our previous proposal that th e B. abortus htrA gene product functions as a stress response protein and further suggest that this protein contributes to virulence. These studies also demonstrate the utility of the broad-host-range plasmid p BBRIMCS for genetic complementation studies in Brucella spp., establis hing a key reagent for more detailed genetic analysis of this importan t zoonotic pathogen.